A team of researchers from Japan, Korea, and the U.S. developed an anti-HIV gene therapy method in which a bacterial gene called mazF is transferred into CD4+ T-cells. The MazF protein is an enzyme (an mRNA interferase) that destroys gene transcripts, preventing protein synthesis. The design of this mazF gene therapy vector ensures that synthesis of the MazF protein is triggered by HIV infection. When HIV infects treated T lymphocytes, MazF is induced, blocking HIV replication and, essentially, making the T-cells HIV resistant.
Transcriptional activation of gene expression directed by the long terminal repeat (LTR) of HIV-1 requires both the transactivation response element (TAR) and Tat protein. HIV-1 mutants lacking a functional tat gene are not able to proliferate. Here we take a genetic approach to suppress HIV-1 replication based on Tat-dependent production of MazF, an ACA-specific endoribonuclease (mRNA interferase) from Escherichia coli. When induced, MazF is known to cause Bak- and NBK-dependent apoptotic cell death in mammalian cells. We first constructed a retroviral vector, in which the mazF (ACA-less) gene was inserted under the control of the HIV-1 LTR, which was then transduced into CD4+ T-lymphoid CEM-SS cells in such a way that, upon HIV-1 infection, the mazF gene is induced to destroy the infecting HIV-1 mRNA, preventing HIV-1 replication. Indeed, when the transduced cells were infected with HIV-1 IIIB, the viral replication was effectively inhibited, as HIV-1 IIIB p24 could not be detected in the culture medium. Consistently, not only cell growth but also the CD4 level was not affected by the infection. These results suggest that the HIV-1-LTR-regulated mazF gene was effectively induced upon HIV-1 IIIB infection, which is sufficient enough to destroy the viral mRNA from the infected HIV-1 IIIB to completely block viral proliferation in the cells, but not to affect normal cell growth. These results indicate that the T cells transduced with the HIV-1-LTR-regulated mazF gene acquire HIV-1 resistance, providing an intriguing potential for the use of the HIV-1-LTR-regulated mazF gene in anti-HIV gene therapy.
RNASE-BASED STRATEGIES for anti-human immunodeficiency virus (HIV) gene therapy may be superior to RNA-based (antisense, ribozyme, or siRNAs) strategies, because the former strategies evade the effects of frequent resistant mutations in HIV-1. MazF is a unique sequence-specific endoribonuclease, or mRNA interferase, encoded by the Escherichia coli genome. It cleaves mRNA at ACA-specific sequences and effectively inhibits protein synthesis. To date, a number of MazF homologues have been found in various bacteria.
In summary, the use of MazF appears to be a novel and highly effective tool for anti-HIV gene therapy. It is effectively able to suppress HIV-1 replication, preventing the emergence of mutated HIV-1. Importantly, MazF induction by invading HIV-1 shows little toxicity to host cells while it efficiently suppresses HIV-1 replication. Specific inhibition of HIV-1 replication by MazF without affecting cell growth is the key feature of MazF-based HIV-1 gene therapy. This may be the first step for RNase-based HIV-1 gene therapy with efficacy in vitro. The feasibility of the MazF-based ex vivo gene therapy may be verified using autologous CD4+ T lymphocytes from HIV-1 patients. To use our mazF vector system for gene therapy, its safety has to be critically evaluated and it should not have any negative impacts on T-cell function. For example, it needs to be shown that there is no alteration in the secretion of functionally important cytokines even though it was observed that MazF expression in HIV-infected CD4+ T cells does not inhibit cell growth. We are currently addressing this question.